The overall goal of the proposed research is to apply static and flow perfusion bioreactor culture of bone sarcoma cells, grown upon a tissue-engineered polymer/extracellular matrix (ECM) hybrid model that reliably mimics key features of the bone tumor niche, to advance our understanding of the IGF-1R/mTOR cancer- related pathway and its clinically-relevant resistance mechanisms. Our laboratory has reported in vivo-like IGF- 1R/mTOR expression patterns, closely related to those observed in human Ewing sarcoma tumors (ES), when established ES cell lines are grown upon innovative biologically inert 3D electrospun poly(?-caprolactone) (PCL) microfiber scaffolds rather than upon traditional plastic monolayers. The present proposal seeks to elucidate the precise mechanisms by which cell-cell and cell-ECM interactions stimulate an activated IGF- 1R/mTOR signaling state within this engineered 3D bone tumor microenvironment, as those interactions are critically important in initiating and maintaining ES. In parallel with determining the influence of those parameters under static cell culture, 3D scaffolds and culture conditions will be adapted to better emulate the native bone microenvironment: (a) varied flow perfusion rates will be used to assess the effect of shear stress upon cell retention while facilitating uniform distribution of Ewing cells within the scaffold, (b) PCL scaffolds will incorporate IGF-1 to mimic the high concentration of IGF-1 naturally released as tumors invade surrounding bone, (c) the effect of an osteogenic ECM upon the IGF-1R/mTOR signaling cascade will be determined using decellularized scaffolds upon which heterotypic mesenchymal stem cells, differentiated toward an osteoblastic lineage, are first grown, and (d) ES cells will be co-cultured with endothelial cells (EC) to determine how heterotypic cells interact within a fabricated 3D bone tumor model to elicit viable ES tumors. Finally, to determine the mechanism(s) by which Ewing sarcoma evades sensitivity to combined mTOR/IGF-1R targeted therapy, freshly-derived tumor specimens (obtained from Ewing sarcoma patients treated with Medi- 573/everolimus in an IRB-approved clinical trial), will be grown in primary culture within 3D scaffolds and compared to 2D culture and patient-derived tumor explants (PDTX) by proteomic analysis of the IGF- 1R/mTOR pathway and putative resistance mechanisms. This novel approach of studying Ewing sarcoma within an ex vivo preclinical model of the bone microenvironment presents tremendous potential for understanding chemotherapeutic efficacy and for determining resistance mechanisms to biologically targeted therapies.